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966 Modulation of the Tumor Suppressor Protein PP2A Using a Small Molecule Agonist Overcomes Multi-Drug Resistance through Mitochondrial Permeability Transition Pore (MPTP) Dependent Induction of Apoptosis in Chronic Lymphocytic Leukemia

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology, Drug Resistance—Lymphoid and Other Diseases: Poster I
Hematology Disease Topics & Pathways:
Leukemia, apoptosis, Diseases, CLL, Non-Biological, Therapies, Non-Hodgkin Lymphoma, Biological Processes, Lymphoid Malignancies, signal transduction
Saturday, December 5, 2020, 7:00 AM-3:30 PM

Kallesh D. Jayappa, PhD1,2, Caroline Farrington, PhD3*, Vicki L. Gordon, BS1,2*, Shekhar Saha, PhD4*, Christopher Morris, BS1,2*, Krista M. Isaac, MD5*, Timothy P. Bender, PhD1,2*, Michael E. Williams, MD6,7*, Craig A. Portell, MD6,8, Goutham Narla, MD, PhD3* and Michael J. Weber, PhD1,2,6*

1Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, VA
2Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA
3Department of Medicine, University of Michigan, Ann Arbor, MI
4Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA
5Division of Hematology/Oncology, University of Virginia Health System, Charlottesville, VA
6Cancer Center, University of Virginia, Charlottesville, VA
7Division of Hematology/Oncology, University of Virginia School of Medicine, Charlottesville, VA
8Division of Hematology/Oncology, University of Virginia, Charlottesville, VA

The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin’s Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017).

Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis.

Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D).

To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C).

In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir.

Disclosures: Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell: Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla: The Icahn School of Medicine at Mount Sinai: Patents & Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.

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